Our computerized database of rat liver epithelial (RLE) cellular polypeptides (Wirth et al., Electrophoresis 1991;12:931-54) has now been expanded to include the analysis of cellular polypeptide alterations during aflatoxin B1 (AFB1)-, spontaneously-, and oncogene (v-Ha-ras, v- raf, and v-myc/v-raf)-induced transformation of RLE cells. Two- dimensional mapping of 35-S-methionine labeled whole cell lysate, cell free in vitro translation products and 32-P-orthophosphate labeled polypeptides revealed subsets of polypeptides specific for each transfor- mation modality. A specific subcellular location for each of the major transformation sensitive proteins was made. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin (TM)- and intermediate filament (IF)-related polypeptides (vimentin, alpha, beta-tubulin, the cytokeratins, and actin) were observed among the various transformant cell lines. Analysis of TM expression in four individual AFB1-, and four spontaneously-induced as well as each of the oncogene transformed cell lines, indicated that five major TM (TM 1-5) isoforms were variably expressed in the various cell lines, including one polypeptide tentatively identified as TM6. Whereas alterations in TM expression appeared to be transformation specific, alterations in the individual IF polypeptides were related more to the differentiation state of the individual cell lines rather than to the transformation phenotype. Our results to date extend our earlier efforts toward the establishment of a comprehensive computerized database of RLE cellular proteins and demonstrates how such a database may serve as a useful resource for studies concerning the regulation of growth and differentiation as well as transformation of RLE cells.